This invention relates to a primer for PCR and also to a method for deciding a base sequence thereof. More particularly, the invention relates to a PCR primer suitable for the adenylation in the course of PCR using a termostable DNA polymerase having terminal transferase activity and also to a method for deciding the sequence of such a PCR primer.
For the detection of DNA or RNA, it is usual to detect after amplification of a sequence of DNA or RNA obtained from a specimen. For this purpose, PCR amplification is usually performed. In order to detect a PCR-amplified DNA fragment, the DNA fragment is subjected to radioisotopic labeling or is labeled with chemical emission or fluorescence, followed by detection of the labeled DNA fragment after separation of a sample such as by gel electrophoresis. Recently, terminal fluorescence labeling by a synthetic DNA and fluorescence labeling by intercalator capable of intercalation with DNA fragments is enabled and has now been in frequent use. When using an intercalating fluorphore, a DNA fragment subjected to electrophoresis with an agarose gel is labeled with an intercalator, such as ethidium bromide, acridine orange or the like, and detected.
For the measurement of a more accurate fragment length, a terminal fluorescence-labeled DNA fragment is subjected to electrophoresis with a polyacyrlamide gel and detected. For a process requiring the measurement of an accurate fragment length, mention is made of RT-PCR (Reverse Transcriptase-PCR, J. Stenman et al.; xe2x80x9cAccurate determination of relative messenger RNA levels by RT-PCRxe2x80x9d Nature Biotechnology, 1999, 17, 720-722), FDD (Fluorescent Differential Display, T. Ito et al.; xe2x80x9cFluorescent differential display: arbitrarily primed RT-PCR finger printing on an automated DNA sequencerxe2x80x9d FEBS Letters, 1994, 351, 231-236), ATAC-PCR (K. Kato; xe2x80x9cAdaptor-tagged competitive-PCR: a novel method for measuring relative gene expressionxe2x80x9d Nucleic Acids Research, 1997, 25, 4694-4696), SSCP (M. Orita et al.; xe2x80x9cDetection of polymorphisms of human DNA by gel electrophoresis as single-Strand conformation polymorphismsxe2x80x9d Proc. Natl. Acad. Sci. USA, 1989, 86, 2766-2770) and the like. In these methods, an amplified product is analyzed with a fluorescent-type DNA sequencer after PCR.
The currently proposed amplification and detection of a DNA fragment based on PCR and electrophoresis has been introduced hereinabove, with many problems undesirably involved in practical applications. More particularly, adenylation to a DNA fragment by the DNA polymerase takes place in the course of PCR, and thus, two peaks are detected based on an adenylated fragment and a non-adenylated fragment, respectively. The probability of the occurrence of the adenylation varies depending on the type of sample DNA and the PCR conditions. This makes it difficult to obtain a peak area of a target DNA fragment due to the splitting of peak.
In SSCP analyses for diagnostic purposes, a peak area is determined for quantitative analysis, enabling one to detect LOH (Loss of Heterozygosity) that will not be judged by a conventional method (K. Sugano et al.; xe2x80x9cSensitive Detection of Loss of Heterozygosity in the TP53 Gene in Pancreatic Adenocarcinoma by Fluorescence-Based Single-Strand Conformation Polymorphism Analysis Using Blunt-End DNA Fragmentsxe2x80x9d Genes, Chomosomes and Cancer, 1996, 15, 157-164, Sensitive Detection of Loss of Heterozygosity in the TP53 Gene in Pancreatic Adenocarcinoma by Fluorescence-Based Singled Strand Conformation Polymorphism Analysis Using Blunt-End DNA Fragments). However, for highly accurate diagnosis, it is necessary to suppress a rate of a peak area of non-adenylated products to peak area of adenylated products to a level within 10%.
To prevent the peak splitting, two procedures are considered including removal of added adenine or positive adenylation caused to occur to 100%. With the method of removal of the added adenine, exnzymatic treatment has to be performed for the removal after PCR (F. Ginot et al.; xe2x80x9cCorrection of some genotyping errors in automated fluorescent microsatellite analysis by enzymatic removal of one base overhangsxe2x80x9d Nucleic Acids Research, 1996, 24, 540-541). On the other hand, in order to cause the adenylation to positively occur, it is necessary to control a concentration of Mg2+ions in a reaction solution and to change reaction conditions. Nevertheless, a difficulty is now involved in stably obtaining an adenylated PCR product. Moreover, although there is a report stating that the efficiency of adenylation changes depending on the sequence in the vicinity of 5xe2x80x2 terminus of a template DNA fragment (V. L. Magnuson et al.; xe2x80x9cSubstrate Nucleotide-Determined Non-Templated Addition of Adenine by Taq DNA Polymerase: Implications for PCR-Based Genotyping and Cloningxe2x80x9d Biotechniques, 1996, 21, 700-709), the sequence is not general, with no decision method of the sequence being proposed.
The method of changing the efficiency of adenylation depending on the sequence at 5xe2x80x2 terminus of a template DNA is called reverse primer tailing (M. J. Brownstein et al.; xe2x80x9cModulation of Non-Templated Nucleotide Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotypingxe2x80x9d Biotechniques, 1996, 20, 1004-1010).
An object of the invention is to design, in reverse primer tailing, a sequence that causes adenylation to generally occur in a high efficiency against any type of PCR primer capable of amplifying a target DNA fragment, thereby providing a primer sequence that can amplify a DNA fragment capable of being simply analyzed by electrophoresis.
To achieve the above object, a PCR primer having an anchor sequence wherein a sequence is designed to cause adenylation to occur in a high efficiency at 5xe2x80x2 terminus of a reverse primer is used according to the invention. The term xe2x80x9canchor sequencexe2x80x9d used herein means a sequence which is positioned at the 5xe2x80x2 terminus of a primer sequence that is complementary with a target gene and which is not complementary with a target DNA sequence. The anchor sequence does not hybridize with a target sequence in the first cycle of PCR, but hybridizes only when complementary strand synthesis proceeds at an opposite strand. Accordingly, the anchor sequence hybridizes in the second and subsequent cycles of the PCR, and the resulting amplified fragment becomes one that has a target sequence and an anchor sequence. The anchor sequence can be designed irrespective of a target DNA sequence, so that it is possible to select a sequence capable of causing adenylation to occur in a high efficiency. It is known that the adenylation of PCR is such that the efficiency of adenylation differs depending on the type of base species at the 5xe2x80x2 terminus, and the efficiency of the adenylation is decided by approximately 5 bases at the 5xe2x80x2 terminus.
In the practice of the invention, four types of primers having anchor sequences wherein only one base at the 5xe2x80x2-terminus among the anchor sequences each made of two to five bases is changed are provided to perform PCR. The results of the PCR are such that the efficiencies of adenylation of the four types of anchor sequences are measured, followed by screening an anchor sequence that is more likely to undergo adenylation. Next, a sequence, in which the adenylation is most likely to occur at the first base of the anchor sequence from the 5xe2x80x2 terminus, is decided, followed by synthesis of four types of primers wherein the second base from the 5xe2x80x2 terminus of the anchor sequence is changed. Like the case of the first base, PCR is performed and a base species with which adenylation is likely to occur at the sequence of the second base from the 5xe2x80x2 terminus is decided. The above procedure is repeated to decide an anchor sequence made of 2 to 5 bases with which adenylation is liable to occur.
PCR is performed using a primer having such an anchor sequence which has been decided by the above procedure and with which adenylation is likely to occur. As a result, an amplified product wherein adenylation has occurred is preferentially obtained. If the ratio of a non-adenylated, amplified product to an adenylated, amplified product is 10% or below, this is usable for diagnosis requiring quantitative analysis.